Abstract
Programmable gene regulators that can modulate the activity of selected targets in trans are a useful tool for probing and manipulating gene function. CRISPR technology provides a convenient method for gene targeting that can also be adapted for multiplexing and other modifications to enable strong regulation by a range of different effectors. We generated a vector toolbox for CRISPR/dCas9-based targeted gene regulation in plants, modified with the previously described MS2 system to amplify the strength of regulation, and using Golden Gate-based cloning to enable rapid vector assembly with a high degree of flexibility in the choice of promoters, effectors and targets. We tested the system using the floral regulator FLOWERING LOCUS T (FT) as a target and a range of different effector domains including the transcriptional activator VP64, the H3K27 acetyltransferase p300 and the H3K9 methyltransferase KRYPTONITE. When transformed into Arabidopsis thaliana, several of the constructs caused altered flowering time phenotypes that were associated with changes in FT expression and/or epigenetic status, thus demonstrating the effectiveness of the system. The MS2-CRISPR/dCas9 system can be used to modulate transcriptional activity and epigenetic status of specific target genes in plants, and provides a versatile tool that can easily be used with different targets and types of regulation for a range of applications.
Highlights
One of the most common approaches for investigating gene function is testing what happens when the gene of interest is misexpressed, usually through transgenic overexpression or through gene knockout or knockdown
We generated all of our MS2-clustered regularly interspaced palindromic repeats (CRISPR)/dead variant of the Cas9 protein (dCas9) expression constructs using the Golden Gatebased GreenGate cloning system [7], which allows rapid assembly of plant binary expression constructs from a library of modular, pre-cloned entry vectors
The ft-10 35S:MS2-VP64/pSUC2:dCas9 T1 plants flowered slightly earlier than ft-10 mutants on average, and the ft-10 pSUC2:MS2-VP64/pSUC2: dCas9 plants slightly later, but overall these plants still closely resembled untransformed ft-10 mutants in their flowering time. These results demonstrate that the MS2-VP64 constructs trigger early flowering through upregulation of the intended target gene FLOWERING LOCUS T (FT), and that the MS2-CRISPR/dCas9 system is an effective method for targeted modulation of gene activity in plants
Summary
One of the most common approaches for investigating gene function is testing what happens when the gene of interest is misexpressed, usually through transgenic overexpression or through gene knockout or knockdown. While these techniques are often very informative, they do have some limitations; for example, they cannot be used to study the impact of epigenetic marks such as DNA methylation or histone modification on individual genes, and it can be difficult to misexpress multiple genes simultaneously, which may be desirable when studying a gene family or a series of steps in a biosynthetic pathway.
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